Two functionally distinct NADP+-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus

J Biol Chem. 2017 Sep 1;292(35):14603-14616. doi: 10.1074/jbc.M117.794172. Epub 2017 Jul 13.


Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP+ oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown.

Keywords: Nfn; archaea; crystal structure; electron bifurcation; elemental sulfur; extreme thermophile; metabolism; nicotinamide.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / isolation & purification
  • Archaeal Proteins / metabolism*
  • Biocatalysis
  • Coenzymes / chemistry
  • Coenzymes / metabolism
  • Crystallography, X-Ray
  • Ferredoxin-NADP Reductase / chemistry
  • Ferredoxin-NADP Reductase / genetics
  • Ferredoxin-NADP Reductase / isolation & purification
  • Ferredoxin-NADP Reductase / metabolism*
  • Ferredoxins / chemistry
  • Ferredoxins / metabolism*
  • Gene Deletion
  • Gene Expression Regulation, Archaeal*
  • Homeostasis
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Models, Molecular*
  • NAD / chemistry
  • NAD / metabolism
  • NADP / chemistry
  • NADP / metabolism*
  • Organisms, Genetically Modified
  • Oxidation-Reduction
  • Phylogeny
  • Protein Multimerization
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / isolation & purification
  • Protein Subunits / metabolism
  • Pyrococcus furiosus / enzymology*
  • Pyrococcus furiosus / genetics
  • Pyrococcus furiosus / growth & development
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism


  • Archaeal Proteins
  • Coenzymes
  • Ferredoxins
  • Isoenzymes
  • Protein Subunits
  • Recombinant Fusion Proteins
  • NAD
  • NADP
  • Ferredoxin-NADP Reductase

Associated data

  • PDB/5JFC
  • PDB/5VJ7